This research will be done primarily in Hungary as an extension of NIH grant # R01 GM62920. Human immunodeficiency virus type 1 (HIV-1) protease is essential for viral replication and has proved an effective target for antiviral drugs to treat AIDS. But, the long term effectiveness of current protease inhibitors as therapeutic agents is limited by the rapid development of drug-resistant variants of the protease. It is necessary to understand the molecular basis for the action of the inhibitor-resistant variants of HIV-1 protease in order to develop new inhibitors and new therapeutic strategies. The aim of the parent proposal is to elucidate the molecular basis for the action of resistant variants of HIV-1 protease by comparing the structures and activities of selected mutants of HIV-1 and Rous sarcoma virus proteases. The long term aim is to predict new protease inhibitors and therapeutic strategies to overcome the problem of drug-resistance. The specificity studies proposed in the parent grant will be extended to cover a broader set of oligopeptide substrates in order to more fully characterize the inhibitor-resistant HIV- 1 protease mutants. Furthermore, new studies on the molecular basis for the specificity of the related human T-cell leukemia virus type 1 (HTLV-1) protease are proposed. These studies will use a combination of kinetic analysis of substrate hydrolysis, mutagenesis, and molecular modeling studies. Understanding the molecular basis of the common characteristics and differences in the specificity of various retroviral proteases including inhibitor resistanl variants of HIV-1 protease will facilitate the design of broad spectrum protease inhibitors. These new inhibitors will have applications in the treatment of AIDS and other human diseases caused by retroviruses.